New technologies are needed to facilitate the analysis, quantitation and imaging of gene expression to exploit the wealth of information being generated, for example, by the Human Genome and Cancer Genome Anatomy Procect. The innovative use of Spliceosome Mediated RNA Trans-splicing (SmaRT) to develop new agents for real time imaging of gene expression within cells, animals, and potentially humans is proposed. Our laboratory has produced different RNAs, known as PTMs (Pre-Trans-splicing Molecules). These PTMs are capable of directing trans-splicing reactions between the PTM and a targeted pre-messenger RNA. The product of a SmaRT reaction is a novel chimeric mRNA, which can encode virtually any desired gene product that may be imaged directly or that can activated or capture a second reporter molecule. The product of a SMaRT reaction contains one or more exons of the target endogenous pre-mRNA and a exonic or cDNA sequence delivered by the PTM. We propose studies to target cancer specific or cancer associated genes: human papillomavirus, human chorionic gonadotropin and EGF receptor with PTMs encoding marker luciferase exons. This application proposes to define the collaboration with the imaging group at the UCLA Medical Center to provide guidance on more advanced imaging techniques and equipment. Our specific aims include the demonstration that SMART can target clinically relevant genes with the luciferase marker at the pre-mRNA level. Subsequent studies will increase the efficiency and specificity of the PTMs through a molecular library. Real time molecular imaging. Real time molecular imaging by spliceosome mediated RNA trans-splicing will greatly facilitate pre-clinical animal studies in cancer gene single copy PCR detection. It may also imaging of tissue distribution of transgenes and vectors, an attractive alternative to single copy PCR detection. It may also be possible to identify metastases in small animal cancer models. If successful these animal studies could lead to human diagnostics of real time cancer specific RNA profiles. SMaRT is attractive single it targets RNA and holds the potential for real time analysis of gene expression.